Problem with RNA isolation/extraction of plasma - When RNA isolation form supernatant it's fine, not from plasma, us (Aug/22/2007 )
I've been 'optimizing' my real time RT-PCR for a while now. Last week when I ran my samples I took along a sample of new virus made which is in cell culture media. Great that I did that, because finally I see that my real time RT-PCR works fine with that sample. The only thing is it doesn't work with all the samples where I am actually designing it for. The only problem I can think of is the samples that don't come up or come up at cycle 38 or so are all from plasma.
More specific mice plasma, we take blood from mice, put it with 10 mM EDTA and do RNA extraction using a Qiagen kit. Which is been working for other people in other labs with pretty much the same samples.
Right now I am planning to do a dilution series of my virus in cell culture media in negative plasma to see if it is inhibiting the RNA extraction. I just was wondering if there were other people that have had this problem or came across something similar. Also, since I am pretty new to the whole real time RT-PCR thing, if you can think of some other experiments that would be great. I would really like to pinpoint this problem some more and hopefully resolve it soon. Since I already am doing a mice experiment and I should already have the assay up and running!
Any help would be very much appreciated. And if you have any questions about how I explained (or failed to explain) the problem!
Hi, we had similar problems and started to do a trizol clean-up first, and then transfer the aqueous phase mixed 1:1 with EtOh 70% onto the Rneasy membrane. That normally worked fine.